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miRNA-431-5p enriched in EVs derived from IFN-β stimulated MSCs potently inhibited ZIKV through CD95 downregulation

Stem Cell Research & Therapy volume 15, Article number: 435 (2024) Cite this article

Abstract

Background

Zika virus (ZIKV) primarily spreads through mosquito bites and can lead to microcephaly in infants and Guillain-Barre syndrome in adults. It is noteworthy that ZIKV can persist in the semen of infected males for extended periods and can be sexually transmitted. Infection with ZIKV has severe pathological manifestations on the testicular tissues of male mice, resulting in reduced sperm motility and fertility. However, there are no approved prophylactic vaccines or therapeutics available to treat Zika virus infection.

Methods

Using a male type I and II interferon receptor-deficient (ifnar1(-/-) ifngr1(-/-)) C57BL/6 (AG6) mouse model infected with ZIKV as a representative model, we evaluated the degree of testicular damage and viral replication in various organs in mice treated with EVs derived from MSC-stimulated with IFN-β (IFNβ-EVs) and treated with controls. We measured testicle size, detected viral load in various organs, and analyzed gene expression to assess treatment efficacy.

Results

Our findings demonstrated that intravenous administration of IFNβ-EVs effectively suppressed ZIKV replication in the testes. Investigation with in-depth RNA sequencing analysis found that IFN-β treatment changed the cargo miRNA of EVs. Notably, miR-431-5p was identified to be significantly enriched in IFNβ-EVs and exhibited potent antiviral activity in vitro. We showed that CD95 was a direct downstream target for miR-431-5p and played a role in facilitating ZIKV replication. miR-431-5p effectively downregulated the expression of CD95 protein, consequently promoted the phosphorylation and nuclear localization of NF-kB, which resulted in the activation of anti-viral status, leading to the suppression of viral replication.

Conclusions

Our study demonstrated that the EVs produced by IFNβ-treated MSCs could effectively convey antiviral activity.

Introduction

Small extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) possess immune regulatory capabilities [1]. As a result, MSC-EVs have emerged as a promising cell-free therapeutic tool with tremendous potential for treating a variety of diseases. Studies have demonstrated that MSCs exhibit enhanced immune regulatory abilities when stimulated by inflammatory factors [2,3,4]. Notably, the presence of IFN-β in MSC culture has been shown to impact the expression of chemokines and their receptors in human MSCs, leading to a significant increase in the secretion of immunomodulatory molecules and their immune modulatory effects [5]. At present, the immunomodulatory and antiviral effects of UC-MSCs are unclear, and UC-MSCs may also have immunomodulatory effects in viral transplacental infection [6]. MSCs treated with interferon can stimulate immune activity [7,8,9], and the antiviral activity of interferon can be transmitted to recipient cells by secreting specific EVs [10]. We found that the enhanced immune function of MSCs induced by IFN-β can be presented by their secreted EVs in line with the evidence that the contents of extracellular vesicles were reflective of the physiological or pathological states of the cells from which they derived [8, 11]. These nanoscale vesicles, composed of natural phospholipid bilayer membranes, can encapsulate various biologically active substances, including nucleic acids (RNA, DNA, non-coding RNA), proteins, and lipids. Through these cargoes, EVs can regulate the physiological functions of distal recipient cells, facilitating the exchange of information between cells and tissues [12].

Zika virus (ZIKV), a flavivirus transmitted by mosquito bites, has caused a number of epidemics in recent years, particularly in the Pacific region, the Caribbean, South and Central America [13]. Infected individuals present a range of symptoms, including fever, body aches, maculopapular rash, and conjunctivitis with severe cases developing Guillain-Barré syndrome [13, 14]. Infection of pregnant women resulted in neonatal microcephaly malformation of the fetus brain, and even stillbirth [15, 16]. Moreover, ZIKV can persistently infect the reproductive systems of male patients, leading to testicular damage in male mice and causing infertility [17,18,19]. The virus can persist in semen for extended periods and can be transmitted through sexual contact [20]. There are currently no approved preventive vaccines or therapeutic drugs available for the treatment of ZIKV infection.

In this study, we isolated EVs from MSCs cultured in the presence of exogenous IFN-β and investigated the virus inhibitory activity of IFNβ-EVs. We found that IFNβ-EVs exhibited enhanced immunomodulatory activity in suppressing virus replication and mitigating virus-induced pathologic manifestations. Through comprehensive in vivo and in vitro analysis, we characterized the antiviral activity of IFNβ-EVs, demonstrating their protective efficacy in male mice against testicular damage induced by infection. To investigate the mechanism of action, EVs composition was analyzed to identify specific antiviral miRNAs encapsulated within IFNβ-EVs and miR-431-5p was identified based on its enrichment in IFNβ-EVs. miR-431-5p exhibited remarkable inhibitory activity on the replication of both ZIKV. While previous studies reported the packaging of miR-431-5p into EVs, enabling its selective enrichment and expression in EVs [21], its antiviral function have not yet been elucidated. Moreover, we unraveled host CD95 as one of the target genes of miR-431-5p and miR-431-5p impeded virus replication by targeting CD95 mRNA and the downregulation of CD95 resulted in the activation of NF-kB, leading to the inhibition of viral replication. The present study provides evidence for the application of cell-free therapy against viral infection.

Materials and methods

Cells and viruses

Human umbilical cord mesenchymal stem cells (HucMSCs) were sourced from GuLou Hospital, an affiliate of Nanjing University, in accordance with the Declaration of Helsinki and approved by the Medical Ethics Committee of Gulou Hospital. These adherent cells were cultivated in low glucose DMEM supplemented with GlutaMAX™, 4 mM L-glutamine, and 10% fetal bovine serum (FBS). Passages 2–8 were used for experiments. Activation of the MSCs was done through the addition of 10 ng/mL of IFN-β to the culture for 48 h. Human cervical carcinoma cell line (Hela) and green monkey kidney cell line (Vero) were kept in a humidified environment with 37℃ and 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Zika virus (SZ01 strain) was propagated in C6/36 cell line, Zika virus strain SZ01/2016 (GenBank: KU866423) had been separated from an ZIKV-infected patient.

Experimental mice

Type I and II interferon receptor-deficient (ifnar1(-/-) ifngr1(-/-)) C57BL/6 (AG6) mice aged 6–8 weeks were procured from the Institute of Zoology, Chinese Academy of Sciences. All procedures involving animals were granted approval by the University of Nanjing Animal Care and Use Committee. Mice were infected with ZIKV by subcutaneous injection (5 to 6 mice per group), and animals were randomly assigned to receive 105 TCID50 units of ZIKV suspended in 100 µL PBS or 100 µL PBS alone. After injection, routine closure was carried out, and the dams were returned to the individual cages for recovery. In addition, depending on the group, mice were injected with different groups of EVs via the tail vein.

For EVs treatment in vivo, mice were anesthetized via the intraperitoneal injection of 1.25 mg/kg of sodium pentobarbital, a well-established anesthetic agent. At the end of the study design point, the animals were euthanized using CO2 infusion at a gas infusion rate of 1.5–3.5 L/min in a box connected to a CO2 line. Once the animals cease respiration for 5–10 min, the euthanasia is confirmed by cervical dislocation. Our reporting of animal experiments adheres to the ARRIVE guidelines 2.0.

Isolation, purification, labeling of EVs

To prepare EVs, cells at 80% confluence were cultured with complete medium supplemented with 10% EVs-depleted FBS for 3 days. Serum EVs were depleted by centrifuging FBS at 120,000 × g for 18 h, effectively eliminating around 95% of FBS extracellular vesicles. Conditioned media from MSC cell cultures, treated with or without IFNs treatment, after centrifuged them for 10 min at 2000 × g to remove any remaining cellular debris, the cleared supernatants were gathered and put into ultracentrifuge tubes (Polyallomer Quick-Seal centrifuge tubes 25 × 89 mm Beckman Coulter) and centrifuged for 30 min at 10,000 × g (Type Ti 45, Beckman Coulter) in an ultracentrifuge (Optima L-90 K or Optima XE-90 Ultracentrifuge, Beckman Coulter) in order to extract macrovesicles. Subsequently, after meticulously gathering the supernatants, a Type 45 Ti rotor was used to centrifuge them for two hours at 110,000 × g and 4 °C. The EVs pellet obtained from this process was reconstituted in PBS and stored at − 80 °C. In addition, the same methodology was used regarding the acquisition of EVs from THP-1 and HEK293T cells, and we have confirmed the obtained information on the characteristics of EVs in other studies (Zou X, et al. Mol Ther; Zhang R, et al. Mol Ther).

Transmission electron microscopy (TEM)

EVs samples (0.2 µg/µL each) were examined using a JEM-2100 transmission electron microscope (JEOL, 100 kV). Samples were fixed to 400 mesh carbon-layered copper grids for up to 2 min. Excess material was drained by blotting, followed by negative staining of samples with 10 µL of uranyl acetate solution (2% w/v; Electron Microscopy Services).

Nanoparticle tracking analysis (NTA)

Size distributions of EVs samples were analyzed using a NanoSight NS300 instrument. Samples were diluted in Milli-Q water and injected via a syringe pump at a flow rate of 50. One-minute videos were captured, and data were obtained in triplicate. Analysis was performed using NTA 3.2 Dev Build 3.2.16 software with auto-analysis settings.

Immunofluorescence assay

Cells from various experimental treatments were fixed with 4% paraformaldehyde for 15–20 min. Subsequently, the fixed cells were perforated in 0.5% Triton X-100 at room temperature for 15 min. The cells were then blocked with 5% serum albumin for a minimum 30 min. Primary antibodies ZIKV Envelope protein antibody (5 µg/mL, GeneTexⓇ, Cat No.GTX133314) and NF-kB p65 antibody (10 µg/mL, proteintechⓇ, Cat No.10745-1-AP), diluted in the incubation solution, were employed to stain the cells overnight at 4 °C. After multiple washes, the cells were incubated with a secondary antibody at room temperature, in the dark for 1 h. The nuclei were then stained with 0.5 µg/mL DAPI, in darkness for 5 min. 10 µl of a mounting agent, infused with an anti-fluorescent quenching agent, was dispensed onto the slide, followed with a sealing procedure. The treated slides were examined by a laser confocal fluorescence microscope (Olympus FluoView FV10i). The resulting images were subjected to thorough analysis and processing, utilizing the system’s integrated software, FV 10i-ASW Viewer 4.0.

Western blots

Using RIPA buffer (Santa Cruz) with a protease inhibitor cocktail (Med ChemExpress), the cultivated cells and ultracentrifuged exosomal pellets were produced, they were then incubated on ice for 30 min. Proteins and the lysates of extracellular vesicles were quantified by BCA (Thermo Fisher Scientific). After that, the samples were subsequently loaded to SDS-PAGE (10% gels) and transferred to PVDF membranes (Millipore). The membranes were blocked for 1 h in 3% BSA and incubated the corresponding antibodies including ZIKV Envelope protein antibody (0.5 µg/mL, GeneTexⓇ, Cat No.GTX133314) and CD95 antibody (0.5 µg/mL, proteintechⓇ, Cat No.13098-1-AP) overnight at 4 °C. Secondary antibodies were IRDye 680RD anti-rabbit immunoglobulin IgG (H + L) and IRDye 800CW anti-mouse IgG (H + L) (Invitrogen). Using an Odyssey Imaging System (LI-COR, Bio-sciences), images were acquired and analyzed.

ELISA assay

IFN-β was detected using commercial ELISA kit (Bio-Techne Valukine™) following the manufacturer’s instructions. Briefly, the naïve-EVs and IFNβ-EVs preparations were added into anti-IFN-β capture antibody-coated ELISA plates, followed by HRP-conjugated secondary antibody (Sigma-Aldrich). The color development was done by addition of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma-Aldrich) at 37oC for 30 min and stopped by 10 µl 0.2 M H2SO4. Optical densities were measured at 450 nm using Infinite 200 (Tecan, Ramsey, MN, USA).

RNA isolation and cDNA synthesis

Total RNAs from cells, EVs, and tissues were extracted using the TRIzol Reagent (Invitrogen, Cat#15596026). To assess mRNA expression, cDNA was synthesized utilizing the PrimeScript™ Reverse Transcriptase (RT) reagent kit (TaKaRa, Cat#RR037A). qPCR for mRNA analysis was conducted utilizing specific primers (Table S1). The mRNA expression levels were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

miRNA or siRNA transfection

MiR-431-5p, miR-18a-3p, miR-380-3p, and siRNA siCD95 were chemically synthesized by RiboBio (China), and working concentration was 20pM. RNA transfections were carried out using the Lipofectamine™ 3000 reagent, following the guidelines provided by the manufacturer. To achieve high efficiency, the transfections were conducted in Opti-medium™ (Gibco, USA). For miRNA analysis, cDNA synthesis was performed using the Bulge-LoopTM miRNA qRT-PCR Starter Kit (RiboBio). qPCR primers for miRNA were procured from RiboBio (Guangzhou, China). The small nuclear RNA U6 was employed as the endogenous control. Real-time qPCR was conducted using SYBR Green kit (Vazyme, China). The 7500 Real-Time PCR System (ABI) was employed for qPCR analysis, and the related primers were displayed in Table S1.

miRNA sequencing analysis

EVs from untreated or IFN-β-treated MSCs were isolated as described above. Microarray analysis of the exosomal miRNA profiles was done by GENEWIZ with the Illumina Novaseq 6000, respectively, with three different EVs preparations. The miRNA-seq data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database with accession number GSE263628.

Luciferase assay

The luciferase reporter plasmids encompassing the wildtype or mutant 3′-UTR of CD95 were prepared by PPL, China. For luciferase assays, HEK-293T cells were seeded at a density of 5 × 104 cells per well in 24-well plates. Following a 24-hour incubation period, the cells were transfected with the luciferase reporter vector, accompanied by the presence of either miRNA NC (miRNC) or miR-431-5p mimic (20 nM), for 24 h. Luciferase activity was quantified utilizing the Diluciferase Reporter Analysis System (Promega) and standardized against Renilla luciferase activity. Each experiment was independently conducted three times.

Statistical analysis

Every data set was displayed with its mean and standard deviation (mean ± S.D.). Data was shown using GraphPad Prism 8.0 software (GraphPad Software, San Diego, California, USA). An analysis of variance was used to compare data for three or more groups (ANOVA), and an independent unpaired Student’s t-test was used to compare data between two groups. A p-value of less than 0.05 indicated a significant difference between the groups.

The work has been reported in line with the ARRIVE guidelines 2.0.

Results

Isolation and characterization of MSC-derived EVs

The ucMSCs were isolated from umbilical cord and characterized by the differentiation potential of the MSCs. The cells could differentiate into osteogenic, adipogenic and chondrogenic in vitro under specific conditions (Figure S1). EVs were purified by multiple ultracentrifugation steps from the conditioned media obtained from MSCs cultured with or without IFNs stimulation for a duration of 2 days [2]. The resulting EVs particles were redissolved in phosphate-buffered saline (PBS) and kept at − 80 °C until further use. The isolated MSC EVs were subjected to comprehensive characterization utilizing various techniques, including Western blot, electron microscopy, and nanoparticle tracking analysis (NTA). Western blot analysis demonstrated that CD9 and Alix, two proteins frequently abundant in EVs, were present in both IFNs-EVs and naive-EVs (Figure S2a). Furthermore, the absence of GM130, a Golgi marker, in the MSC EVs pellet indicated the absence of contamination by vesicles generated from the endoplasmic reticulum (Figure S2a). Transmission electron microscopy (TEM) analysis revealed that the EVs preparation had a distinctive spherical form and fell within the anticipated size range (Figure S2b). NTA analysis indicated an average diameter of approximately 119 nm (naive-EVs), 111 nm (IFNα-EVs),110 nm (IFNβ-EVs) and 111 nm (IFNγ-EVs) (Figure S2c). Collectively, these findings demonstrated that IFN-β treatment did not alter the basic characteristics of EVs from MSC. Moreover, by immunofluorescence analysis, we found that the uptake efficiency by recipient cells was similar for EVs derived from both sources (Figure S2d).

IFNβ-EVs strongly suppressed virus replication

To assess the roles of MSCs-derived EVs on viral replication, EVs were prepared from MSCs treated with the same concentration of IFN-β, IFN-α or IFN-γ and EVs from un-treated MSCs (naive-EVs) that served as a comparison (Fig. 1a). Purified naive-EVs, IFNα-EVs, IFNβ-EVs, or IFNγ-EVs were subsequently added to ZIKV-infected Vero cells and the un-infected cells simultaneously, the use of the Vero cell culture model was due to the susceptibility to ZIKV infection. 48 h after infection, total protein extraction was performed for Western blot analysis. The expression levels of ZIKV’s envelope protein and prM indicated a significant inhibition of virus replication in cells treated with IFNβ-EVs but markedly less effect by IFNα-EVs (Fig. 1b). IFNβ-EVs was able to reduce the viral protein by almost 70% as compared with the mock and the naive-EVs treatment groups (∗∗∗∗p < 0.0001) (Fig. 1c). Viral genomic RNA analysis further corroborated the observations (Fig. 1d). Similar results were observed in human testicular Sertoli cells, and the significant antiviral effect of IFNβ-EVs was again confirmed (Figure S3a-d). Consistently, immunofluorescence staining of the viral envelope showed that infected Vero cells treated with various EVs exhibited the most pronounced reduction of the viral envelope expression in cells treated with IFNβ-EVs but less by IFNα-EVs (Fig. 1e), as shown in Fig. 1b, suggesting that viral replication was inhibited most effectively by IFNβ-EVs. In contrast, IFNγ-EVs facilitated viral replication as evidenced by the increased envelope expression, viral genomic RNA (Fig. 1b-d). The expression of viral proteins at different time post infection in the cells treated with 50 µg/mL IFNβ-EVs also confirmed that IFNβ-EVs had a strong inhibitory effect on viral replication (Fig. 1g, Figure S4). We also showed that IFNβ-EVs inhibition of ZIKV replication was dose-dependent, as shown by both Western blot analysis of viral Envelope (Fig. 1h-i) and qPCR analysis of vRNA (Fig. 1j). To ensure that the purified IFNβ-EVs did not contain carry-over IFN-β, we treated Vero cells with IFNβ-EVs and analyzed the ISG15 activation by measuring mRNA levels. As being defective in endogenous type I interferon, Vero cells respond to exogenous IFN-β stimulation, resulting in the upregulation of ISG15 expression [22]. No significant increase of ISG15 mRNA was observed by IFNβ-EVs treatment (Figure S5). Additionally, direct ELISA analysis confirmed the absence of free IFN-β in IFNβ-EVs (Fig. 1k). These evidences suggest that the viral inhibition by IFNβ-EVs was not mediated by the carry-over IFN-β. To rule out the potential cell specific effect, the viral inhibitory activity of IFNβ-EVs was also investigated on HeLa cells, and viral inhibitory activity was confirmed as evidenced by Western blot and qPCR (Figure S6a-b). Notably, the level of CXCL10 mRNA in HeLa cells did not exhibit significant changes (Figure S6c), while there was a slight alteration in ISG15 mRNA levels, possibly due to varying degrees of natural immune response induced by different viral replication (Figure S6d). ZIKV-infected Vero cells were also treated with EVs derived from THP-1 cells or 293T cells pre-treated with IFN-β. Interestingly, the observed virus-inhibiting effect was unique to MSCs-derived EVs, whereas the EVs derived from IFNβ-treated THP-1 cells or 293T cells did not exhibit antiviral activity (Figure S7). Collectively, these observations suggest that the anti-viral activity of IFNβ-EVs was unique to MSCs and not mediated by carry-over IFN-β.

Fig. 1
figure 1

IFNβ-EVs suppressed virus replication in vitro. (a) Schematic presentation of IFNβ-EVs suppressed virus replication in vitro experiment. (b-c) Vero E6 cell samples from different treatment groups were collected, western blot analysis of envelope and prM protein being specific proteins for ZIKV, GAPDH as the reference protein. (d) Quantitative RT-PCR analysis of ZIKV genomic RNA in the different treatment groups. (e-f) Immunofluorescence staining images of the cells of envelope (in red), and DAPI (in blue) (scale bar: 20 μm). (g) Western blot analysis of ZIKV replication at different time in Vero E6 cells cultured with or without IFNβ-EVs (50 µg/mL). (h-i) Western blot analysis of ZIKV (MOI = 1) replication in Vero E6 cells cultured with different doses of IFNβ-EVs. (j) Quantitative RT-PCR analysis of viral genomic RNA expression in ZIKV-infected Vero cells treated with different doses of IFNβ-EVs. (k) Detection of free IFN-β (n = 3) in IFNβ-EVs prepared by differential centrifugation using the ELISA method. All data were expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA and multiple comparative trials of Bonferroni (**p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Fig. 5

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IFNβ-EVs reduced viral load in ZIKV-infected mice and alleviated testicular injury

In vivo experiments were conducted in 6-week-old type I interferon receptor deficient male mice [AG6, ifnar1(-/-) ifngr1(-/-)] since wild-type mice do not exhibit prominent symptoms after ZIKV infection and clear the virus quickly [23]. AG6 mice exhibit noticeable symptoms such as impaired mobility, fetal microcephaly, testicular shrinkage, and potential mortality and have been widely used as an in vivo model for ZIKV infection [23]. As outlined in Fig. 2a, mice were administered 50 µg IFNβ-EVs one day prior to inoculation of 103 PFU ZIKV, followed with injections of 50 µg IFNβ-EVs each day post infection and at every other day for 8 days. After 14 days of infection, groups of mice were killed and dissected, and viral load in serum and various organs is determined by qRT-PCR. The experimental animals also consisted of a PBS control group and a naive-EVs treatment group, which served as a control. Throughout the experiment, mice in each group showed a slight decrease in body weight for ZIKV-infected mice, as compared with the mice treated with PBS only, while the animals treated with naive-EVs and IFNβ-EVs exhibited much less reduction in weight than those of the infected mice without treatment (Figure S8). At the end of the 18-day experimental period, ZIKV-infected mice were anesthetized and euthanized, and their tissues were collected for subsequent analysis. Notably, male AG6 mice infected with ZIKV exhibited testicular damage and significant shrinkage, as shown in Fig. 2b and d. In contrast, mice treated with IFNβ-EVs or naive-EVs did not display significant changes in testicular size (Fig. 2b). Surprisingly, the testes of mice treated with naive-EVs did not show significant signs of atrophy, indicating that naive-EVs possess the ability to inhibit viral replication in vivo through their immunomodulatory properties, consistent with previous descriptions of naive-EVs [24,25,26]. In Fig. 2C, DiR-labeled EVs can be enriched in the testes through the blood-testicular barrier, shows the fluorescence enrichment at 1/2/4/24 h post-injection. Viral copy numbers in the testicles, brain, blood, liver, kidney, and spleen tissues showed a significant reduction in mice treated with IFNβ-EVs as compared to the mice without the EVs treatment. Additionally, the viral load in the tissues of mice treated with naive-EVs was also significantly reduced, albeit with significantly less reduction as compared to IFNβ-EVs (Fig. 2e-i).

Fig. 2
figure 2

IFNβ-EVs treatment can reduce viral load in ZIKV-infected mice and alleviate symptoms of testicular injury caused by viral infection. (a) Schematic representation of the procedure to infect ZIKV and the treatment (naive-EVs or IFNβ-EVs) that the mice received at day 14. (b) Photographs of testicular tissue from each treatment group were taken to retain the image (scale bar is shown in the figure). (c) The distribution of the EVs in a mouse delivered by tail vein injection was monitored in real time during a 4-h period using an IVIS (Xenogen). Each mouse was injected with 100 µg of DiR-labeled EVs (above). Exosome distribution to organs in the mice (below). (d) HE stained tissues, including testicular tissue of mice from various treatment groups (scale bars: 100 μm and 50 μm), the red arrow indicated the location of the testicular injury. (e-i) The total RNA of the tissue was extracted, and the virus copy in the testes (e), brain (f), liver (g), kidneys (h), and spleen (i) was detected by qPCR after reverse transcription. All data were expressed as mean ± standard deviation (SD) (n = 4–5) and were analyzed by one-way ANOVA and multiple comparative trials of Bonferroni (*p < 0.05, **p < 0.01, ****p < 0.0001)

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Mir-431-5p enriched in IFNβ-EVs exhibited a significant inhibitory activity against ZIKV replication

The primary constituents of EVs include a variety of proteins, lipids, and RNAs. However, due to the limited abundance of proteins and lipids, and the amplified cascade effects of miRNAs, we selected miRNA as the focus of our research. Through sequencing and analysis of differentially expressed miRNAs in IFNβ-EVs and naive-EVs, we identified a range of miRNAs that were enriched in IFNβ-EVs, among which miR-431-5p, etc., were enriched in IFNβ-EVs (Fig. 3a-b), and not enriched in IFNα-EVs (Figure S9). Through the miRDB website, we screened the genes that miR-431-5p may target, and analyzed the signaling pathways enriched by KEGG, most of which are related to viral replication (Fig. 3c). With miR-431-5p being enriched by more than 40-fold as compared to that in naive-EVs (Fig. 3d), whereas the expression of miR-431-5p at cellular level was not affected by IFN-β treatment of MSCs (Fig. 3e), suggesting that miR-431-5p was likely selectively enriched. To investigate the roles of enriched miRNAs in IFNβ-EVs on viral replication, we selected miR-431-5p, miR-3615, miR-18a-3p, and miR-380-3p, based on their higher IFNβ-EVs levels, for analysis by transfecting HeLa cells (which are easier to transfect and more susceptible to ZIKV’s infection) with analogues of these three miRNAs and subsequently infecting the cells with ZIKV (Fig. 3f, S10a). The overexpression of miR-431-5p and miR-380-3p significantly inhibited ZIKV replication, as evidenced by a significant reduction in viral envelope protein expression (Fig. 3f-i). miR-18a-3p had only a minor inhibitory effect on virus replication, as compared to the other two miRNAs (Fig. 3f, S10a-b), whereas miR-3615 showed no inhibitory effect on viral replication (Figure S11). Additionally, miR-431-5p demonstrated a dose-dependent inhibition of viral replication (Fig. 3j-l). These findings establish the antiviral roles of these miRNAs encapsulated in IFNβ-EVs, particularly miR-431-5p.

Fig. 3
figure 3

miR-431-5p, enriched in IFNβ-EVs, had a significant effect on inhibit ZIKV replication. (a) Naive-EVs and IFNβ-EVs were sequenced and analyzed to screen for differentially expressed miRNAs. (b) Data show the log fold-change of up-regulated miRNA. (c) KEGG enrichment analysis of miR-431-5p target genes. (d) Quantitative RT-PCR analysis of miR-431-5p in the Naive-EVs and IFNβ-EVs by miRNA-specific primers. (e) Quantitative RT-PCR analysis of miR-431-5p in the treatment with IFN-β at different working concentrations. (f) Western blot analysis of Envelope protein expression in HeLa cells transfected with miR-431-5p, miR-18a-3p, miR-380-3p mimics, ZIKV infection (MOI = 1) was performed, miRNCs were the control experimental group. (g) Extraction of HeLa cells infected with ZIKV after transfection of miR-431-5p mimics, reverse transcription by miRNA-specific primers, and detection of miR-431-5p expression by qPCR. (h-i) After RNA reverse transcription extracted from the above cells, qPCR detects viral genome and envelope protein mRNA expression. miRNCs were the control experimental group. (j) Western blot analysis of Envelope protein expression in HeLa cells transfected with miR-431-5p mimics (20, 40, 80 pM). (k-l) Quantitative RT-PCR analysis of miR-431-5p and ZIKV RNA in HeLa cells transfected with miR-431-5p mimics (20, 40, 80 pM). All data were expressed as mean ± standard deviation (SD) (n = 3) and were analyzed by one-way ANOVA and multiple comparative trials of Bonferroni (**p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Fig. 6.

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Mir-431-5p targeted CD95 of the host and inhibited its translation

miRNAs modulate biological processes by targeting mRNAs and either directly degrading target mRNAs or influencing their stability, ultimately inhibiting their translation. To investigate the mechanisms of miR-431-5p in inhibiting ZIKV replication, we, through analyzing the miRDB database for gene list queries, identified that the cell surface death receptor, CD95, is a target of miR-431-5p and identified three sequences within the 3’ UTR region of CD95 that are complementary to the miR-431-5p seed region sequence (Fig. 4a). To confirm that CD95 mRNA is indeed the target of miR-431-5p, we transfected exogenous miR-431-5p mimics into HeLa cells and observed a dose-dependent downregulation of CD95 protein expression (Fig. 4b), while mRNA levels remained unaffected (Fig. 4c). Furthermore, we constructed a plasmid vector containing two-segment sequences of the CD95 3’ UTR region and using a double luciferase reporter system as depicted (Fig. 4a), and confirmed direct binding between miR-431-5p and the CD95 3’ UTR. The luciferase activity of CD95 gene was significantly reduced in the presence of miR-431-5p mimics compared to the negative control (miRNC) group (Fig. 4d). However, no difference in luciferase activity was observed when the binding sites were mutated (Fig. 4d), suggesting that miR-431-5p-induced downregulation of CD95 was sequence specific. Given that AGO2 plays a crucial role in RISC (RNA-induced silencing complex)-mediated miRNA degradation and enrichment, we performed RIP analysis [27, 28] of the cells transfected with miR-431-5p mimics using anti-AGO2 antibody and detected both miR-431-5p and CD95 mRNA (Fig. 4e), suggesting that both miR-143-5p and CD95 mRNA were associated with AGO2. Together, these experiments confirmed CD95 as the target for miR-431-5p.

Fig. 4
figure 4

miR-431-5p targetd the CD95 mRNA 3’UTR region and effectively affectd its translation expression. (a) Schematic diagram of miR-431-5p and CD95 3’UTR region complementary fragments. (b) Western blot analysis of CD95 in HeLa cells transfected with miR-431-5p mimics or negative control (miRNC). (c) Quantitative RT-PCR analysis of CD95 mRNA in HeLa cells transfected with miR-431-5p mimics or negative control (miRNC). (d) Construct a double luciferase fluorescent reporter plasmid with a complementary sequence of miR-431-5p in the CD95 3’UTR region and a plasmid at the mutation site, co-transfer the plasmid with miR-431-5p into 293T cells, collect cell lysate for detection after 24 h, and the miRNC group was a negative control (n = 4). (e) RIP assay using AGO2 antibody to detect miR-431-5p and CD95 mRNA expression, miRNC group was a negative control. All data were expressed as mean ± standard deviation (SD) (n = 3) and were analyzed by one-way ANOVA and multiple comparative trials of Bonferroni (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Fig. 7

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ZIKV replication was inhibited by reduced CD95 expression

CD95 plays crucial roles in regulating inflammation of HSV infection [29, 30]. To investigate the roles of endogenous CD95 on ZIKV replication, we examined CD95 expression over time in ZIKV-infected HeLa cells and observed that CD95 expression increased from 24 hpi when the viral envelope protein became detectable (Fig. 5a-c). In addition, CD95 expression was significantly correlated with increasing MOIs of ZIKV (Fig. 5d-e). These observations indicated that CD95 was beneficial to ZIKV replication. CD95 was also significantly upregulated in the testes and brain tissues of ZIKV-infected mice (Fig. 5f-g). The expression of CD95 mRNA in the testes and brains of ZIKV-infected mice treated with IFNβ-EVs was significantly lower than that in the ZIKV-infected group (Fig. 5h-i). Interestingly, knockdown of CD95 by siCD95 resulted in noticeable decrease in the expression of the ZIKV envelope protein in HeLa cells, as shown by Western blot analysis (Fig. 5j). Consistent with the western blot results, viral genomic RNA was significantly reduced when endogenous CD95 expression was downregulated by siCD95 (Fig. 5k-l). These observations suggest that CD95 expression was required for ZIKV replication.

Fig. 5
figure 5

Decreased endogenous CD95 expression affectd ZIKV replication. (a-c) HeLa cells were infected with ZIKV (MOI = 1) for 0–60 h. Whole cell lysate and total RNA were collected for detection by western blot analysis and quantitative RT-PCR analysis, to detected the expression of CD95 and ZIKV envelope. (d-e) Quantitative RT-PCR analysis of CD95 and envelope in HeLa cells were infected with ZIKV (MOI = 0,1,2,4,8,16,32) for 48 h. (f-g) AG6 mice were infected with ZIKV, quantitative RT-PCR analysis of CD95 mRNA expression in testis and brain. (h-i) AG6 mice from various treatment groups, quantitative RT-PCR analysis of CD95 mRNA expression in testis and brain. (j-l) HeLa cells transfected with siCD95 (40, 80 pM) for 24 h or negative control (miRNC), then infected with ZIKV (MOI = 1) for 48 h. Western blot analysis of CD95 and ZIKV envelope in HeLa cells. Quantitative RT-PCR analysis of CD95 (k) and viral genomic RNA (l) in HeLa cells. All data were expressed as mean ± standard deviation (SD) (n = 3) and analyzed by one-way ANOVA and multiple comparative trials of Bonferroni. All data were taken from the mean ± standard deviation (SD) of the three parallel experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Fig. 8

Full size image

Mir-431-5p affected phosphorylation of NF-kB and the expression of cytokines by targeting CD95

CD95 was reported to play a role in NF-kB activation [31]. To illustrate the mechanisms of action, we examined the effects of miR-431-5p overexpression on NF-kB in HeLa cells and observed that p65 phosphorylation was significantly increased in HeLa cells overexpressing miR-431-5p, indicating its activation of the NF-kB (Fig. 6a). Immunofluorescence showed nuclear translocation of NF-kB upon treatment with miR-431-5p or siCD95 (Fig. 6b). Immunoblotting analysis of HeLa cells transfected with either miR-431-5p or siCD95 showed elevated p65 phosphorylation, further supporting NF-kB activation (Fig. 6c). To assess the downstream effects of reduced CD95 expression on NF-kB-mediated immune responses, we measured the mRNA levels of IL-6, IL-1β, CXCL2, CXCL10, and TNF-α in HeLa cells treated with siCD95. siCD95 treatment of ZIKV-infected Hela cells significantly elevated mRNA levels of IL-6, IL-1β, CXCL2, CXCL10, and TNF-α, the major downstream pro-inflammatory effectors of NF-kB activation (Fig. 6d). These results suggest that either miR-431-5p upregulation or downregulation of CD95 expression led to the activation of NF-kB signaling pathway, as evidenced by increased p65 phosphorylation and nuclear translocation, resulting in the activation of innate immune response against the ZIKV replication (Fig. 6e).

Fig. 6
figure 6

miR-431-5p activated the NF-kB pathway in tumor cells by targeting CD95. (a) Western blot analysis of the phosphorylation level of P65 NF-kB in HeLa cells transfected with miR-431-5p (working concentration of 80 pM). (b) Fluorescence in situ hybridization analysis of P65 NF-kB (green) in Hela cells tranfected with siCD95 and miR-431-5p (80 pM). Nuclei were stained with DAPI (blue). The scale bar is 5 μm. (c) Western blot analysis of the phosphorylation level of P65 NF-kB in HeLa cells transfected with siCD95 (40 pM) and miR-431-5p (40 pM and 80 pM). (d) Quantitative RT-PCR analysis of IL-6, IL-1β, CXCL2, CXCL10, TNF-α, CCL2 mRNA level in ZIKV-infected HeLa cells before being transfected with siCD95 (80 pM). (e) Quantitative RT-PCR analysis of IL-6, CXCL2, CXCL10, TNF-α, CCL2 mRNA level in ZIKV-infected HeLa cells before being transfected with miR-431-5p (80 pM). All data were expressed as mean ± standard deviation (SD) (n = 3) and analysed by one-way ANOVA and Bonferroni’s multiple comparative test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Full-length blots are presented in Supplementary Fig. 9

Full size image

Discussion

Extracellular vesicles (EVs) derived from mesenchymal stem cells have received widespread attention in recent years for their reparative and therapeutic roles on diseases. Previous studies showed the reparative functions of MSC-derived EVs closely associated with their actions in immune and inflammatory regulations [32, 33]. EVs derived from MSC could function in the cure of disease or immune modulation through regulatory RNAs such as miRNAs [34] or through delivery of functional protein molecules [35].

Although clinical studies involving the use of ucMSC or MSC-derived ΕVs have been extensively carried out in a variety of diseases, there are only limited studies on the antiviral effects of MSC-EVs. Our data suggest that IFNβ-MSC-EVs exhibited potent antiviral activity against ZIKV, as demonstrated by the reduction of viral gene expression and viral titers in both cell culture and mouse models of infection. miR-431-5p, one of the miRNAs highly enriched in IFNβ-EVs, exhibited antiviral effects by targeting the host protein CD95 to promote the activation of NF-kB and thus the release of downstream cytokines. One of the reasons that IFNβ-EVs exerted stronger antiviral activity is that IFN-β treatment of MSC allowed the enrichment of immune stimulatory miRNAs, such as miR-431-5p, in the EVs. Since the functional roles of miR-431-5p in animals will require significant sets of experiments that will be beyond the scope of the present study, we did not further investigate the presence and abundance of miR-431-5p in animal experiment, as well as the antiviral mechanisms of CD95, NF-kB and downstream pro-inflammatory effectors (IL-6, IL-1β, CXCL2, CXCL10, and TNF-α) involved, and further studies will be carried out on the antiviral effects and mechanisms of miR-431-5p in vivo.

Previous studies have demonstrated that the functional mechanism of mesenchymal stem cells (MSCs) rely heavily on paracrine signaling, with small EVs potentially playing a crucial role in this process [1, 36,37,38]. Notably, stem cells possess immunomodulatory capabilities, and previous experiments have revealed similar functionality in their EVs [36]. Type I interferons (IFNs) are known to play a critical role in inducing antiviral molecules and inhibiting early viral infections of host cells [39, 40]. Moreover, researches indicated that EVs could facilitate the transfer of interferon-mediated antiviral function between cells [10, 41]. For instance, IFN-α was shown to stimulate the release and uptake of EVs, enabling the transfer of antiviral molecules from liver non-parenchymal cells to hepatocytes, suggesting that IFN-α could directly inhibit viral replication in infected cells while indirectly impeding viral transmission by transferring antiviral molecules via EVs to uninfected cells [10, 42]. Additionally, EVs have the capacity to transport IFN-α-associated miRNAs from macrophages to hepatitis B virus (HBV)-infected hepatocytes, thereby exerting anti-viral activity against HBV replication. IFNs was reported to impair exosomal secretion by inducing protein ISGylation [43] and IFN-α/β were found to increase the expression of hACE2 on the surface of EVs, which suppressed SARS-CoV-2 replication in vitro and in vitro [44]. These studies indicate that interferons can modulate the content of EVs secreted and influence the biological activity of the EVs. Our results revealed a significant bias in the sorting of target miRNAs into MSC-derived EVs after IFN-β stimulation, though the level of those miRNAs was similar in cytoplasm before and after the treatment. However, miR-431-5p is clearly not the only miRNA in the MSC-EVs responsible for the anti-viral activity since non-IFN-β stimulated MSC-EVs exhibited in vivo viral inhibitory activity too, albeit at a reduced level. Non-specific antiviral effects of stem cell EVs have also been reported in the past, and the mechanism may be attributed to their immunomodulatory ability [45, 46]. Notably, this IFNβ-induced antiviral activity was not observed in other cell types such as 293T or THP1, indicating that the interferon induced selective packaging of miRNA might be a specific property associated with MSCs.

Previous studies have pointed out the existence of some miRNAs with antiviral activity [47]. miRNAs can target viral transcripts or cellular factors involved in viral replication, suppressing viral multiplication or enhancing host defense mechanisms [48, 49]. CD95, previously recognized as a cell death receptor for its roles in apoptosis induced by members of the tumor necrosis factor (TNF) receptor superfamily [50, 51], was recently found to regulate non-apoptosis-related pathways [30, 52, 53]. During viral infection, host cells upregulated CD95 expression without undergoing apoptosis, indicating that CD95’s role in viral infection is beyond simply mediating apoptotic pathways [29]. Furthermore, the CD95 homologous ligand, CD95L, is associated with immune homeostasis and immune surveillance, and mutations in CD95 and CD95L have been implicated in autoimmune diseases such as systemic lupus erythematosus (SLE) and cancer [54]. Recent research has unveiled that CD95 functions as both a death receptor, inducing apoptosis through binding to membrane CD95L, and an activator of non-apoptotic pathways, promoting inflammatory responses and tumor growth upon binding to soluble CD95L [53, 55]. Notably, a recent study indicated that CD95/Fas suppresses NF-kB activation [31]. Although virus infections can upregulate CD95 expression, there is a lack of previous research on the consequences of downregulating CD95 expression on viruses. Current research on the mechanism of ZIKV infection of the testicular system is limited, and no definitive conclusions have been drawn on the damage of ZIKV to the testicular system [56]. It is uncertain whether human Sertoli cells can represent the testicular system when infected with ZIKV in vitro. Therefore, this limits the study of the mechanism of CD95 during ZIKV replication in the testis. We showed that downregulation of CD95 expression led to the activation of the NF-kB signaling pathway, upregulating pro-inflammatory cytokines and chemokines to mediate immune responses against ZIKV infection. Clearly, the ZIKV-induced upregulation of CD95 suppressed the antiviral innate immunity, which is probably a virus counteract to the host’s CD95-activated apoptosis responding to the infection. However, we cannot rule out that in addition to miR-431-5p and CD95, EVs derived from stem cell may also have other mechanisms of immune conditioning and antiviral factors.

Numerous studies found that EVs derived from MSCs were capable of inhibiting the activation of NF-kB, especially when the EVs acted on macrophages, leading to the attenuation of inflammation [57,58,59,60]. Studies showed that the administration of MSCs-EVs downregulated the expression of NF-kB p65 and reduced the production of NO, IL-1β, and IL-18 in colonic macrophages, alleviating colitis [61]. Further studied suggested that microRNA-146a (miR-146a) was an anti-inflammatory miRNA that acted as a negative feedback regulator of colonic macrophages in MSC-EV-based relief of intestinal inflammation [57]. The anti-inflammatory effects of MSC-derived EVs were mediated by cellular factors such as TGF-β [62] and HLA-G563 or by miRNAs such as miR-132、miR-2134,64,65. In our study, we discovered that miR-431-5p was selectively enriched in EVs derived from MSC treated with IFN-β and induced NF-kB activation through targeting at cellular CD95, thereby suppressing viral replication in host cells. To our best knowledge, miR-431-5p has not been reported to possess antiviral activity and its downregulation of cellular CD95, resulting in the activation of antiviral status is a novel observation. According to the results obtained in this study, the antiviral effect of miR-431-5p is achieved by targeting CD95, indirectly affecting the activation of NF-kB and affecting the expression of downstream pro-inflammatory effectors. However, the antiviral effect of miR-431-5p may not be limited to this approach, so more in-depth research is needed on the antiviral mechanism of miR-431-5p.

Our study demonstrated that EVs derived from MSCs can alleviate symptoms in the male reproductive system caused by ZIKV infection, addressing an unmet clinical need as no vaccine or medicine is available at the present [20]. Utilizing EVs as therapeutic vehicles and drug delivery systems offers several potential advantages over traditional drug delivery methods such as liposomes and nanoparticles: their decreased size lessens the possibility that they may become trapped in tiny capillaries following systemic infusion, which could enhance the delivery of medication to the illness site; being cell-free, EVs have a longer shelf life, fewer side effects, and lower risks compared to cells; Immunomodulating capability of MSC-derived EVs expands the potential of EVs as therapeutic tools, as the synergistic action of complex mixtures of factors targeting different therapeutic pathways may enhance therapeutic efficacy through individual factors. The natural cellular origin of MSC-EVs and its therapeutic contents present better biocompatibility, lower immunogenicity and superior biosafety.

Conclusions

In conclusion, our study enhances the understanding of the underlying mechanisms by which MSCs contribute to the treatment of viral infections. Our research opens up the possibility of using IFN-induced MSCs for the effective treatment of ZIKV infection and its associated reproductive system lesions.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. miRNA-seq data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database with accession number GSE263628.

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Acknowledgements

We would like to thank the State Key Laboratory of Analytical Chemistry for Life Science for assistance with the experiment.

Funding

This work was supported by grants from National Natural Science Foundation of China (NSFC) (No.81672020, 82072423, 82272502, 31970149, and U22A20335), the Nation Key Research and Development Program of China (2023YFB3810200, 2023YFB3810204).

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Authors and Affiliations

  1. Center for Public Health Research, Medical School, Nanjing University, Nanjing, 210093, China

    Meng Yuan, Xiaoyan Tian, Wenyuan Ma, Nan Zheng & Zhiwei Wu

  2. Department of Clinical Medicine, Medical School of Nanjing University , Nanjing, 210093, China

    Yu Jin

  3. Nanjing Children’s Hospital, Nanjing Medical University, Nanjing, People’s Republic of China

    Yu Jin

  4. State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, People’s Republic of China

    Zhiwei Wu

  5. Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing, People’s Republic of China

    Zhiwei Wu

  6. Department of Orthopedics, Northern Jiangsu People’s Hospital, Clinical Teaching Hospital of Medical School,Nanjing University, Yangzhou, China

    Yongxiang Wang

  7. Department of Infectious Diseases, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, PR China

    Rui Zhang

  8. Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, 43205, USA

    Xue Zou

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Contributions

Meng Yuan, Wenyuan Ma: conceptualization; data curation, validation and analysis; software; writing of original draft; Rui Zhang, Xiaoyan Tian, Xue Zou: data curation and validation; visualization; Zhiwei Wu, Yongxiang Wang, Nan Zheng, Yu Jin: project administration and supervision; writing, review and editing, funding acquisition and resources. All authors read the article and approved the submitted version.

Corresponding authors

Correspondence to Yu Jin, Nan Zheng, Zhiwei Wu or Yongxiang Wang.

Ethics declarations

Ethics approval and consent to participate

All animal experimental protocols were approved by the Nanjing University Animal Care Committee and followed the ‘Guide for the Care and Use of Laboratory Animals’ published by the Chinese National Institutes of Health. And the approval number is No: IACUC-D2202180, the date of approval was January 2023. The research protocols were conducted in accordance with the animal behavioral guidelines, using approved protocols from the institutional animal care committee. For umbilical cord mesenchymal stem cells (HucMSCs) collection, the study entitled “Stem cells are extracted from clinical patient samples (tissues, blood, body fluids) and aborted fetal tissues for basic regenerative medicine and clinical disease treatment research” was approved by the Clinical Stem Cell Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School (Date: 11. 29. 2023, No.2017-161-08) and was conducted following approved institutional guidelines. Written informed consent was obtained from all donors prior to this study.

Competing interests

The authors declare that they have no competing interests. The authors declare that artificial intelligence is not used in this study.

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Yuan, M., Tian, X., Ma, W. et al. miRNA-431-5p enriched in EVs derived from IFN-β stimulated MSCs potently inhibited ZIKV through CD95 downregulation. Stem Cell Res Ther 15, 435 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04040-4

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512